Plastic and Reconstructive Surgery • March 2014 Supplement
Saturday, March 8, 2014 50 Fabrication of Tissue-Engineered Human Constructs for Patient Specific Auricles Rachel C Hooper, MD1; Rachel Nordberg, BA2; Kadria N Derrick, MD1; Jennifer Puetzer, BA3; Karina A Hernandez, DO1; Ope Asanbe, MD1; Larry Bonassar, PhD3; Jason A Spector, MD, FACS1 1 Weill Cornell Medical College, New York, NY, 2Cornell Univeristy, Ithaca, NY, 3Cornell University, Ithaca, NY Purpose: The reconstruction of pediatric microtia using autologous donor cartilage is limited by significant obligatory donor site, pain and scarring as well as frequent suboptimal aesthetic outcome. Tissue-engineering allows for the creation of anatomically correct auricular constructs and the minimization or even elimination of the previously mentioned complications. In previous work, we fabricated patient specific, high fidelity tissue-engineered frameworks composed of type I collagen and bovine auricular chondrocytes that not only maintained shape and size over 12 weeks, but also exhibited proteoglycan and elastin deposition, with mechanical properties indistinguishable from native auricular cartilage. As a bridge to clinical translation, we have now synthesized human auricular chondrocyte (HAC) constructs in order to determine the optimal chrondrocyte passage and seeding density for the fabrication of patient specific tissue-engineered auricles. Methods: Human auricular cartilage was obtained from discarded specimens following elective office otoplasty procedures; chondrocytes were extracted and expanded. Type 1 collagen (10 mg/ml) was seeded with 25million cells/mL HAC, passage 2–3 and subsequently underwent thermal gelation. After formation of a 1 mm thick sheet, 8mm diameter scaffolds were obtained using a biopsy punch. Scaffolds were stored in Dulbeco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 μg/mL penicillin, 100 μg/mL streptomycin, 0.1 mM non-essential amino acids for 48 hours prior to surgical implantation. Scaffolds were implanted subcutaneously in the dorsa of 8-week-old nu/nu mice and harvested after 1 month. Following harvest, tissues were processed for imaging and histology. Results: Gross inspection of HAC scaffolds following 1 month of implantation demonstrated maintenance of scaffold size and shape. Post-harvest confocal reflectance microscopy revealed viable chondrocytes within the collagen matrix. Picrosirus red staining demonstrated the presence of lacunar chondrocytes with local deposition of cartilage whereas Verhoeff staining demonstrated elaboration of elastin fibers within the construct.
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Conclusion: We have successfully fabricated viable human auricular chondrocyte constructs that form a cartilaginous matrix and elaborate elastin fibers in as little as 1 month after implantation. Similar techniques are currently being explored to determine the optimal passage and seeding density to allow for the generation of 250 million auricular chondrocytes necessary to seed a full-sized ear scaffold.
Saturday, March 8, 2014 50 Fabrication of Tissue-Engineered Human Constructs for Patient Specific Auricles Rachel C Hooper, MD1; Rachel Nordberg, BA2; Kadria N Derrick, MD1; Jennifer Puetzer, BA3; Karina A Hernandez, DO1; Ope Asanbe, MD1; Larry Bonassar, PhD3; Jason A Spector, MD, FACS1 1 Weill Cornell Medical College, New York, NY, 2Cornell Univeristy, Ithaca, NY, 3Cornell University, Ithaca, NY Purpose: The reconstruction of pediatric microtia using autologous donor cartilage is limited by significant obligatory donor site, pain and scarring as well as frequent suboptimal aesthetic outcome. Tissue-engineering allows for the creation of anatomically correct auricular constructs and the minimization or even elimination of the previously mentioned complications. In previous work, we fabricated patient specific, high fidelity tissue-engineered frameworks composed of type I collagen and bovine auricular chondrocytes that not only maintained shape and size over 12 weeks, but also exhibited proteoglycan and elastin deposition, with mechanical properties indistinguishable from native auricular cartilage. As a bridge to clinical translation, we have now synthesized human auricular chondrocyte (HAC) constructs in order to determine the optimal chrondrocyte passage and seeding density for the fabrication of patient specific tissue-engineered auricles. Methods: Human auricular cartilage was obtained from discarded specimens following elective office otoplasty procedures; chondrocytes were extracted and expanded. Type 1 collagen (10 mg/ml) was seeded with 25million cells/mL HAC, passage 2–3 and subsequently underwent thermal gelation. After formation of a 1 mm thick sheet, 8mm diameter scaffolds were obtained using a biopsy punch. Scaffolds were stored in Dulbeco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 μg/mL penicillin, 100 μg/mL streptomycin, 0.1 mM non-essential amino acids for 48 hours prior to surgical implantation. Scaffolds were implanted subcutaneously in the dorsa of 8-week-old nu/nu mice and harvested after 1 month. Following harvest, tissues were processed for imaging and histology. Results: Gross inspection of HAC scaffolds following 1 month of implantation demonstrated maintenance of scaffold size and shape. Post-harvest confocal reflectance microscopy revealed viable chondrocytes within the collagen matrix. Picrosirus red staining demonstrated the presence of lacunar chondrocytes with local deposition of cartilage whereas Verhoeff staining demonstrated elaboration of elastin fibers within the construct.
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Conclusion: We have successfully fabricated viable human auricular chondrocyte constructs that form a cartilaginous matrix and elaborate elastin fibers in as little as 1 month after implantation. Similar techniques are currently being explored to determine the optimal passage and seeding density to allow for the generation of 250 million auricular chondrocytes necessary to seed a full-sized ear scaffold.
